Abstract
Barnacles are the only sessile lineages among crustaceans, and their sessile life begins with the settlement of swimming larvae (cyprids) and the formation of protective shells. These processes are crucial for adaptation to a sessile lifestyle, but the underlying molecular mechanisms remain poorly understood. While investigating these mechanisms in the acorn barnacle, Amphibalanus amphitrite, we discovered a new gene, bcs-6, which is involved in the energy metabolism of cyprid settlement and originated from a transposon by acquiring the promoter and cis-regulatory element. Unlike mollusks, the barnacle shell comprises alternate layers of chitin and calcite and requires another new gene, bsf, which generates silk-like fibers that efficiently bind chitin and aggregate calcite in the aquatic environment. Our findings highlight the importance of exploring new genes in unique adaptative scenarios, and the results will provide important insights into gene origin and material development.
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Data availability
The A. amphitrite genome and all of the sequence data have been deposited with BioProject under accession: PRJNA878644. The genome assembly and annotation files are available at figshare (https://doi.org/10.6084/m9.figshare.21310305)105. RNA-seq data of differential developmental stages were deposited with BioProject under accession: PRJNA877841. The induced and inhibited settlement of barnacles RNA-seq were deposited in the SRA database under accession: PRJNA878556. The mass spectrometry proteomics data have been deposited in the iProX integrated proteome resources under the accessions: PXD039109 and PXD039111. Source data are provided with this paper.
Code availability
The custom pipelines and scripts were deposited at Zenodo106 and GitHub (https://github.com/ZhaofangHan/Acorn-Barnacle-Genome)107.
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Acknowledgements
We thank Y. Wu for assistance with sample collection, Y. Zou for help with data submission and G. Duan and M. Han for their support in making the schematic drawings of barnacles, all from Xiamen University. Z.F. acknowledges funding from the National Natural Science Foundation of China (32102775). Y.-Y.Z. acknowledges funding from the National Natural Science Foundation of China (32071485). D.F. acknowledges funding from the National Natural Science Foundation of China (42376090), the National Key Research and Development Program of China (2022YFC3106004 and 2022YFC3103904), the Project of Fujian Ocean Synergy Alliance (FOCAL2023-0302), the Science and Technology Project of Fujian Province (2022H0003) and the MEL-RLAB Program (202101).
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D.F., C.K., Y.-Y.Z. and Z. Han conceived and designed the scientific objectives. Z.W. and P.S. collected samples for sequencing DNA and RNA. Z. Han assembled the barnacle genomes and contributed to all of the data analyses. Z.W. conducted settlement experiments and performed the RNA pull-down, ISH, EMSA and Dual-Luciferase reporter assay and analyzed the resulting data. Z. Han conducted an analysis of the transcriptomic and proteomic data of shell. Z.W. performed SEM, I-SEM and Raman microscopy to validate shell structures. Z.W. performed in vivo RNAi of bcs-6 and in vitro crystallization and binding assays to validate bsf functions. Z. Huang, L.C., H.H., S.Y. and M.H. provided suggestions for the data analysis. Z. Han, Z.W., and Y.-Y.Z. drafted the first version of the manuscript and D.F., C.K., Y.-Y.Z., Z. Han, Z.W. and D.R. contributed to the revision of the manuscript.
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Extended data
Extended Data Fig. 1 Genome survey and Hi-C interaction heatmap of Amphibalanus amphitrite.
a, Genome size was estimated by dividing the total number of effective k-mers by the number of homo-peaks. Genome-wide heterozygosity rate and repetitive content were estimated by GenomeScope using the k-mer 17 histogram. b, The upper panel shows the Hi-C interaction heatmap of A. amphitrite across all chromosomes. The genome was split into 100 kb bins, and the colors represent the interaction frequencies between individual chromosomes. The lower panel shows an example of a region on Chr11. Below the heatmap is a view of the first eigenvector (PC1) used to determine the A (positive) and B (negative) compartments, the directionality index (DI) used to call topologically associating domains (TADs) at a 40kb resolution, and gene density of the reference sequence (RefSeq). The black arrows in the TAD triangular map indicate boundaries. c, Size distribution of topological domains (TADs) and their boundaries (d), and gene densities in different chromatin domains (e).
Extended Data Fig. 2 Comparative genomic analysis of barnacles and other arthropods.
a, The distribution of divergence of transposable elements (TEs) of eight crustaceans. SINE/LINE: short/long interspersed nuclear element; LTR: long terminal repeat; DNA: DNA transposon. b, The expanded (red) and contracted (blue) gene families, and the frequency distribution of synonymous substitution rates (Ks) between pairwise paralogues for nine crustaceans and the outlier species. c, The 604 expanded families in the common ancestor of barnacles were filtered, where families with a total gene count across these species of less than five were excluded, resulting in 26 gene families. The top nine gene families are listed in Fig. 1d. Abbreviations: Grik2, glutamate receptor ionotropic, kainate 2; mGluR, metabotropic glutamate receptor; nAChRs, nicotinic acetylcholine receptors; Tuba, tubulin alpha chain; Tubb, tubulin beta chain; FT, cadherin-related tumor suppressor; STS, steryl-sulfatase; SPR, sex peptide receptor; Tmem2, transmembrane protein 2; Setd1, histone-lysine N-methyltransferase; Tret1, facilitated trehalose transporter; FucTC, alpha-(1,3)-fucosyltransferase C; WFDC3, WAP four-disulfide core domain protein 3.
Extended Data Fig. 3 Loss of Hox3 and abd-A in barnacle genomes.
a, TAD structures around Hox genes at a 40 kb resolution. The anterior genes (lab, pb, Dfd, Scr, ftz and Antp) and posterior genes (Ubx and Abd-B) were grouped into two TADs. The black arrows stand for TAD boundaries. b, Alignments of short and long reads to genomic regions of the two absent homeobox genes (Hox3 and abd-A). c, Numbers of transposable elements (TEs) enriched across the homeobox gene region. d, The alignment of Hox3 homeodomain (represented in red) in six arthropod species, namely, Parhyale hawaiensis, Litopenaeus vannamei, Portunus trituberculatus, Eriocheir sinensis, Daphnia pulex and Strigamia maritima.
Extended Data Fig. 4 Transcriptomic analysis of settled and unsettled samples in experiments I and II.
a, Sample legends of the two experiments. b, Cluster and PCA analysis of samples, and GO enrichment of differentially expressed genes (DEGs) for experiment II. The color scale indicates the dissimilarity between pairwise samples in gene expression profiles, and a darker color indicates that the gene expression profiles are more similar. One replicate was discarded due to being an outlier. If the replicate were included in the analysis, there would be limited consistent results between the two experiments, that is, many fewer overlapping terms. Only significantly enriched terms are displayed in the right panel. c, Cluster and PCA of samples, and GO enrichment of DEGs for experiment I. d, Venn diagram of DEGs in the two experiments, and Gene Ontology (GO) analysis results for the 91 overlapping DEGs between the two experiments. e, Weighted gene coexpression network analysis (WGCNA) of experiment I. The upper panel on the left displays a cluster analysis based on the dissimilarity between pairwise gene expression patterns of experiment I. The upper panel on the right shows the eigengene expression pattern of the Blue Module. The eigengene represents the gene expression profiles of a module. The lower panel illustrates the correlation between the eigengene expression pattern and phenotypes (settled vs. non-settled) for different modules, with the Blue Module exhibiting the highest correlation. In b, c and d, the P values in enrichments analysis were calculated using the hypergeometric test and adjusted for multiple comparisons with clusterProfiler. Padj < 0.05 and q value < 0.05 were considered significant enrichments.
Extended Data Fig. 5 Domain structures and sequences of proteins involved in cyprid settlement, and immunofluorescence analysis results for TGase.
a, The nine genes encode proteins that contain Tryp_SPc (trypsin) domains. The protein domains were predicted using a web server (http://pfam-legacy.xfam.org). b, The sequence alignment of Tryp_SPc domains of Homo sapiens thrombin and nine proteins. c, Ribbon drawings showing the structural similarity between Tryp_SPc domains of H. sapiens thrombin and that of nine proteins. Note that the comparison between the Tryp_SPc domain of one gene (AaSB) and H. sapiens thrombin has already been shown in Fig. 2e. d, Ribbon drawings showing the structural similarity between A. amphitrite transglutaminase (TGase, AlphaFold identifier: AF-A0A6A4WL68-F1) and different H. sapiens TGase. TGase1 (PDB: 1GGT) and TGase3 (PDB: 1L9M). e, Negative control of immunofluorescence analysis of TGase expression in the attachment disc of an A. amphitrite juvenile (representative images of n = 2 independent experiments).
Extended Data Fig. 6 Alignment of bcs-6 transposon in A. amphitrite genome, and the location and expression of bcs-6 in P. pollicipes genome.
a, Sequence alignments of the U3 region in the 5′ LTR of 26 intact bcs-6 gene copies in the A. amphitrite genome. Cis-regulatory elements (CREs) were predicated by the web server AliBaba2.1. b, The locations of three intact bcs-6 gene copies and their expression levels in the P. pollicipes genome. The expression levels were calculated with published RNA-seq data (NCBI, no. PRJNA394196) from pooled larvae and adults of P. pollicipes and visualized by a tool in Integrative Genomics Viewer (IGV). c, The schematic diagrams of shedding cyprid shells of an acorn barnacle and a stalked barnacle, modified from video-based observations under the Creative Commons CC-BY-NC license40.
Extended Data Fig. 7 Function analysis of A. amphitrite bcs-6.
a, The visualization of siRNA in the knockdown experiment after treating cyprids with control agents and cy3-labeled siRNA for 12 hours (representative images of n = 2 independent experiments). NC-1: negative control, 2 μL ml−1 LipoHigh; NC-2: negative control, cy3-labeled siRNA against gfp; siRNA-1: cy3-labeled siRNA-1 against bcs-6; siRNA-2: cy3-labeled siRNA-2 against bcs-6. Red channel signals were extracted to reveal the precise interfering location of siRNA in the cyprids. b, Fluorescence in situ hybridization (FISH) analysis revealed subcellular localization of overexpressed bcs-6 (the experiment was performed once). c, KEGG enrichment of co-expressed genes with bcs-6 across different developmental stages. d, SDS-PAGE and silver staining of the RNA pull-down eluents (representative images of n = 2 independent experiments). e, Transcriptional pattern of 24 adjacent genes within a range of 10 kb upstream and downstream of bcs-6, among which five exhibited a coexpression pattern with bcs-6 (shown in red).
Extended Data Fig. 8 Chemical and proteomic analysis of barnacle shell plates.
a, The Raman spectrum was employed to determine the types of crystals produced by in vitro mineralization. Mineralization was conducted in CaCl2 solution, with the addition of BSA or BSF (protein products of rctg452.10). b, The Venn plot of proteomic analysis, including three technical replicates per plate type. c, Transcriptional levels of all of the genes identified in the nine expanded gene families with functions related to shell formation (Fig. 1d).
Extended Data Fig. 9 Predicted protein dimer of ctg452.10, and motif comparison of ctg452.10 and ctg452.9.
a, The predicted structure of the ctg452.10 protein dimer was generated using HDOCK software (http://hdock.phys.hust.edu.cn/). The presence of lysine (K) and glutamine (Q) in the ctg452.10 protein suggests the potential formation of amide bonds. b, The motifs of proteins encoded by ctg452.10 and ctg452.9 in A. amphitrite, as well as the corresponding homologous sequence in P. pollicipes, were predicted using MEME (https://meme-suite.org). c, The predicted motifs were aligned using ClustalW for sequence comparison.
Supplementary information
Supplementary Information
Supplementary Notes 1–3 and Supplementary Tables 1–17.
Supplementary Data
Supplementary Data 1: The mass spectrometry of shell proteomics data. Supplementary Data 2: The mass spectrometry of bcs-6-binding proteins in the RNA pull-down assay.
Source data
Source Data Figs. 1–5 and Extended Data Figs. 1–9
Statistical source data.
Source Data Fig. 2
Barnacle photos and immunofluorescence.
Source Data Fig. 3
EMSA gel and whole-mount in situ hybridization.
Source Data Fig. 4
Microscopy scans and immuno-electron microscopy (IEM).
Source Data Fig. 5
Microscopy scans, coomassie staining gel and binding blot.
Source Data Extended Data Fig. 5
Immunofluorescence images.
Source Data Extended Data Fig. 7
FISH and RNA pull-down gel.
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Han, Z., Wang, Z., Rittschof, D. et al. New genes helped acorn barnacles adapt to a sessile lifestyle. Nat Genet (2024). https://doi.org/10.1038/s41588-024-01733-7
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DOI: https://doi.org/10.1038/s41588-024-01733-7